Expression of Protective Antigens in Transgenic Chloroplasts and the Production of Improved Vaccines

ABSTRACT

Vaccines for conferring immunity in mammals to infective pathogens are provided, as well as vectors and methods for plastid transformation of plants to produce protective antigens and vaccines for oral delivery. The invention further provides transformed plastids having the ability to survive selection in both the light and the dark, at different developmental stages by using genes coding for two different enzymes capable of detoxifying the same selectable marker, driven by regulatory signals that are functional in proplastids as well as in mature chloroplasts. The invention utilizes antibiotic-free selectable markers to provide edible vaccines for conferring immunity to a mammal against  Bacillus anthracis , as well as  Yersina pestis . The vaccines are operative by parenteral administration as well. The invention also extends to the transformed plants, plant parts, and seeds and progeny thereof. The invention is applicable to monocot and dicot plants.

RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 12/014,352filed Jan. 15, 2008, which is a continuation of U.S. Ser. No. 10/500,351filed Jan. 3, 2005, now issued as U.S. Pat. No. 7,354,760, which is anational stage filing of PCT/US02/41503 filed Dec. 26, 2002, whichclaims priority to U.S. Ser. Nos. 60/400,816 filed Aug. 2, 2002,60/393,651 filed Jul. 3, 2002, and 60/344,704 filed Dec. 26, 2001 whichare incorporated herein in their entirety by reference.

BACKGROUND

Yersinia pestis is the causative agent of bubonic and pneumonic plague.Bacillus anthracis is the causative agent for the anthrax disease. TheCenters for Disease Control and Prevention (hereinafter “CDC”) lists Y.pestis and B. anthracis as two of the six Category A biological agentsthat pose a risk to national security.

Yersinia pestis

The etiologic agent of plague is the Gram-negative bacterium Yersiniapestis. The natural route of transmission of Y. pestis from one animalhost to another is either directly or via a flea vector. Plague isendemic in some regions of the world and outbreaks occasionally occur asa consequence of natural disasters. Y. pestis is also a concern as oneof the microorganisms with potential for use against civilian ormilitary populations as a biological warfare/biological terrorism agent.In such a situation, the pneumonic form of plague would be the mostlikely outcome. This form of plague is particularly devastating becauseof the rapidity of onset, the high mortality, and the rapid spread ofthe disease. Immunization against aerosolized plague presents aparticular challenge for vaccine developers. There is currently novaccine for plague.

Both live attenuated and killed plague vaccines have been used in man,although questions remain about their safety and relative efficacy,especially against the pneumonic form of infection. Since plague remainsendemic in some regions of the world, and because of the possibility ofthe illegitimate use of Y. pestis as a biological warfare agent,development of improved vaccines against plague is a high priority. Theideal vaccine should be deliverable in a minimum of doses and quicklyproduce high titer and long-lasting antibodies. Moreover, such a vaccineshould protect against aerosolized transmission of Y. pestis.

The two most recently described approaches to development of improvedplague vaccines are 1) attenuated mutants of Y. pestis and 2) subunitvaccines. The potential efficacy of attenuated mutants of Y. pestis asvaccines is supported by experience with the live attenuated vaccinestrain EV76. This vaccine has been in use since 1908 and is given as asingle dose Immunization of mice with EV76 induces an immune responseand protects mice against subcutaneous and inhalation (aerosolized)infection. However, this vaccine strain is not avirulent and has anunacceptable safety profile. Moreover, multiple variants of theclassical EV76 strain exist that differ significantly in passage historyand genetic characteristics. Recent studies have focused on creatingdefined genetically attenuated mutants of Y. pestis, similar to thosecreated in other Gram-negative bacteria (i.e., Salmonella spp.). Forunknown reasons, genetic mutations, which markedly attenuate Salmonellaspp. do not attenuate Y. pestis. For instance, an aroA mutant of Y.pestis was fully virulent in the murine model of disease but avirulentin guinea pigs.

A number of potential subunit vaccines have been evaluated forimmunogenicity and protective efficacy against Y. pestis. The two mostpromising are F1 and V. F1 is a capsular protein located on the surfaceof the bacterium and the V antigen is a component of the Y. pestis TypeIII secretion system. These proteins have been produced recombinantlyand induce protective immune responses when administered individually. Acombination or fusion of F1 and V may have an additive protective effectwhen used to immunize humans against plague. It is thought that F1-Vfusion protein should provide protection against both subcutaneous andaerosol challenge, and will have the potential to provide protectiveimmunity against pneumonic as well as bubonic plague due to either wildtype F1⁺ Y. pestis or to naturally occurring F1-variants. To date no onehas been able to express the F1-V fusion protein in transgenicchloroplast. Such an accomplishment would provide a large supply ofhigh-quality antigen for vaccines.

Bacillus anthracis

Bacillus anthracis is the organism that causes the anthrax disease. Itis a Gram-positive, nonmotile, aerobic or facultatively anaerobic,spore-forming bacterium. The spores are about 1 .mu.m in size, extremelyhardy, resistant to gamma rays, UV light, drying, heat, and manydisinfectants. Spores germinate upon entering an environment rich inglucose, amino acids, and nucleosides, such as in animal and humantissues and blood. The vegetative cells enter the spore state when thenutrients are exhausted or when the organisms are exposed to molecularoxygen in the air.

Anthrax is typically a disease of animals, especially herbivores such ascows, sheep, and goats. It affects humans through contact with thespores in one of three ways. Cutaneous anthrax occurs when the sporesenter the body through a cut or an abrasion on the skin.Gastrointestinal anthrax occurs when the spores enters the body throughconsumption of contaminated meat products. Inhalation anthrax occurswhen the spores enter the body through inhalation of the spores.

When spores enter the body, macrophages engulf them, migrate to regionallymph nodes and the spores germinate into vegetative bacteria.Macrophages release the vegetative bacteria and they spread through theblood and lymph until there are up to 10.sup.8 bacilli per milliliter ofblood. The exotoxins are produced from bacteria and they lead tosymptoms and possible death. Spores can survive in the lungs or lymphnodes up to 60 days before germination occurs. In animal experiments, ithas been seen that once toxin secretion has reached a criticalthreshold, death will occur, even if the blood is rendered sterilethrough the use of antibiotics. From primate studies, the estimatedlethal dose of inhaled anthrax spores sufficient to kill 50% of humansexposed to it (the LD50) is 2,500-55,000 spores.

The CDC lists anthrax as a category A disease agent and estimates thecost of an anthrax attack would be $26.2 billion per 100,000 personsexposed. The only vaccine licensed for human use in the U.S., Biothrax(formerly Anthrax vaccine adsorbed, or AVA), is an aluminumhydroxide-adsorbed, formalin-treated culture supernatant of a toxigenic,nonencapsulated, non-proteolytic strain of Bacillus anthracis. Inaddition to the immunogenic protective antigen (PA), the vaccinecontains trace amounts of edema factor (EF) and lethal factor (LF) thatmay contribute to the local reactions seen in 5-7% of vaccinerecipients, or reported to be toxic causing side-effects. There is aclear need and urgency for an improved vaccine for anthrax and forimproved production methods that allow it to be mass-produced atreasonable cost.

There are two main virulence factors associated with B. anthracis, thepolyglutamyl capsule which is believed to prevent the vegetativebacterial cells from being phagocytized and the exotoxins. Two differentexotoxins are produced by three factors. PA binds to the host cell, LFis a zinc metalloprotease which inactivates mitogen-activated proteinkinase. The edema toxin is formed when PA binds to EF. This toxinincreases cyclic AMP (cAMP) levels in the cell which upsets the waterhomeostasis resulting in accumulation of fluid called edema. The lethaltoxin is formed from binding of PA and LF. This toxin stimulatesmacrophages to release interleukin-1b, tumor necrosis factor a, andother cytokines which contribute to shock and sudden death.

Anthrax has become a serious threat due to its potential use inbioterrorism and recent outbreaks among wild-life in the United States.Concerns regarding vaccine purity, the current requirement for sixinjections followed by yearly boosters, and a limited supply of the keyprotective antigen (PA), underscore the urgent need for an improvedvaccine.

SUMMARY OF THE INVENTION

The present invention pertains to vaccines for conferring immunity inmammals to infective pathogens, as well as vectors and methods forplastid transformation of plants to produce protective antigens andvaccines for oral delivery. The invention further provides transformedplastids having the ability to survive selection in both the light andthe dark, at different developmental stages by using genes coding fortwo different enzymes capable of detoxifying the same selectable marker,driven by regulatory signals that are functional in proplastids as wellas in mature chloroplasts. The invention utilizes antibiotic-freeselectable markers to provide edible vaccines for conferring immunity toa mammal against Bacillus anthracis, as well as Yersina pestis. Thevaccines are operative by parenteral administration as well. Theinvention also extends to the transformed plants, plant parts, and seedsand progeny thereof. The invention is applicable to monocot and dicotplants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic view of anthrax chloroplast constructs accordingto the present invention, and a view of a southern blot. FIG. 1A showsthe pLD-JW1 vector used for chloroplast transformation. FIG. 1B showsthe pLD-JW2 construct. FIG. 1C shows PCR with the primers 3P and 3M.FIG. 1D shows a PCR analysis of randomly selected clones.

FIG. 2 is a schematic view of anthrax chloroplast constructs accordingto the present invention, and views of southern blots. FIG. 2A showsexpected products from digestion of wild type untransformed plant. FIG.2B shows expected products from digestion of a plant transformed withpLD-JW1. FIG. 2C shows expected products from digestion of a planttransformed with pLD-JW2. FIG. 2D shows a flanking sequence probeshowing heteroplasmy in pLD-JW1 line and homoplasmy in pLD-JW2 lines.FIG. 2E shows a pag sequence probe showing presence of pag in transgeniclines.

FIG. 3 shows Western blots demonstrating PA expression of transgeniclines containing different constructs. FIG. 3A shows a western blot ofpLD-JW1 T.sub.1 transgenic lines for PA quantification. Lane 1: wildtype; Lane 2: Ladder; Lane 3: 20 ng PA; Lane 4: 10 ng PA; Lane 5: 5 ngPA; Lane 6: 1:10 dilution pLD-JW1 line; Lane 7: 1:20 dilution pLD-JW1line. FIG. 3B shows a western blot of pLD-JW2 T.sub.1 transgenic linesfor PA quantification. Lane 1: wild type; Lane 2: Ladder; Lane 3: 20 ngPA; Lane 4: 10 ng PA; Lane 5: 5 ng PA; Lane 6: blank; Lane 7: 1:10dilution pLD-JW2 line #1; Lane 8: 1:20 dilution pLD-JW2 line #1; Lane 9:1:10 dilution pLD-JW2 line #2; Lane 10:1:20 dilution pLD-JW2 line #2.FIG. 3C shows a western blot comparing extraction buffers, containingCHAPS and SDS, and both, and measuring stability of extracts at4.degree. C., where “Sup” means supernatant fraction, “Hom” meanshomogenate (soluble and insoluble fractions). The construct used waspLD-JW2.

FIG. 4 shows western blots of T.sub.1 pLD-JW1 plant in continuous lightin FIGS. 4A and 4B, and in FIG. 4C shows histogram of .mu.g PA/g freshtissue in young, mature, and old leaves after 3 and 5 days of continuousillumination.

FIG. 5 shows graphs of the results of macrophage cytotoxic assays forextracts from transgenic plants. FIG. 5A shows supernatant andhomogenate samples from T.sub.0 pLD-JW1 tested. FIG. 5B showsSupernatant samples from T.sub.1 pLD-JW1 tested.

FIG. 6 is a schematic view of tomato vector construct pLD Tom-BADH.

FIG. 7 is a schematic of the pTOM-BADH2-G10-pag tomato vector constructFIG. 8 shows PCR analysis of the products of tomato vectors using BADHprimers, wherein + is pTOM-G10-PA vector control, − is WT Tomato plant,and #3 is transgenic tomato plant.

FIG. 9 shows tomato seedlings 12 days after seed germination.

FIG. 10 shows tomato cotyledons ready for bombardment.

FIG. 11 shows cut and bombarded cotyledons.

FIG. 12 shows the serum anti-PA as determined by ELISA and the in vitrotoxin neutralization by serum antibodies following intranasalimmunization.

FIG. 13A is a schematic view of a pLDS-F1V vector construct, while FIG.13B shows restriction enzyme analysis of the pLDS-F1V vector.

FIG. 14A shows PCR reactions to determine pLDS-F1V vector integrationinto the chloroplast genome of Petit Havana, while 14B shows a secondPCR reaction, which is also used to determine pLDS-F1V transgeneintegration.

FIG. 15A shows a Western Blot of pLDS-F1V from XL1-Blue strain of Ecoli, while 15B shows Western Blots of F1V expression in transgenicchloroplasts.

FIG. 16 is a schematic of constructs to be inserted in edible plants,where “gene X” represents each of LF27-PA63, CTB-LF27,LF27-PA63+CTB-LF27, LF27+PA.

FIG. 17A is a schematic view of a pDD34-ZM-gfp-BADH vector construct,while 17B shows the subsequent expression of the construct containingGFP in E. coli.

FIG. 18A is a schmetactic of pDD33-ZM-aadA-BADH vector construction, and18B shows the subsequent expression of the construct in E. coli grown onspectinomycin.

FIG. 19(A-C) show GFP expression in embryogenic maize cultures where ais non-transgenic control and b and c are transformed maize embryoniccalli.

FIG. 20A shows maize control and transgenic plants on regenerationmedium containing spectomycin, while FIG. 20B shows PCR confirmation ofchloroplast transgenic plants using appropriate primers.

FIG. 21A is a schematic view of a pDD37-DC-gfp-BADH carrot chloroplasttransformation vector, while 21B illustrates GFP expression in E. coli.

FIG. 22A is a schematic of a vector construct pDD36-DC aadA-BADH carrotchloroplast transformation vector, while 22B illustrates the expressionof the vector using E. coli cells grown on spectinomycin.

FIG. 23(A-D) shows Expression of GFP in different stages of transgeniccultures of carrot.

FIG. 24 is a schematic view of a Double Barreled Plastid Vectorharboring aphA-6 and aphA-2 genes conferring resistance toaminoglycosides according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION Chloroplast Engineering

The concept of chloroplast genetic engineering, allows the introductionof isolated intact chloroplasts into protoplasts and regeneration oftransgenic plants. Early investigations involving chloroplasttransformation focused on the development of in organello systems usingintact chloroplasts capable of efficient and prolonged transcription,translation, and expression of foreign genes in isolated chloroplasts.However, the discovery of the gene gun as a transformation device madeit possible to transform plant chloroplasts without the use of isolatedplastids and protoplasts. Chloroplast genetic engineering has beenaccomplished in several phases. Studies have been made on the transientexpression of foreign genes in plastids of monocots and dicots. Uniqueto the chloroplast genetic engineering is the development of a foreigngene expression system using autonomously replicating chloroplastexpression vectors. Stable integration of a selectable marker gene intothe tobacco chloroplast genome was also accomplished using the gene gun.Recently, useful genes conferring valuable traits via chloroplastgenetic engineering have been demonstrated. Plants resistant to Bacillusthuringiensis (Bt) sensitive and resistant insects were obtained byintegrating the cryIAc and cry2A genes into the tobacco chloroplastgenome.

Chloroplast genomes of plants have also been genetically engineered toconfer herbicide resistance where the introduced foreign genes werematernally inherited. This was a significant step in the development ofcommercially viable genetically modified plants because it alleviatesany concerns over the problem of out-crossing traits with weeds or othercrops.

For large-scale foreign protein production, plants are an ideal choicedue to the relative ease of genetic manipulation, rapid scale up(million seeds per plant), large biomass, and the potential to findalternative uses for various crops. A remarkable feature of chloroplastgenetic engineering is the observation of exceptionally largeaccumulation of foreign proteins in transgenic plants (as much as 46% ofCRY protein in total soluble protein) even in bleached old leaves. Usingchloroplast transformation technology, large quantities of protectiveantigen can be produced in transgenic plants, due to the presence ofthousands of copies of transgenes per cell as opposed to only a fewcopies in nuclear transgenic plants. By “protective antigen” is meantthat the antigen elicits an immunogenic response in mammals whenadministered by an appropriate route in an appropriate amount.Transgenic chloroplast technology has been used to hyper-expressbacterial proteins—up to 46% of total soluble protein from Bacillusgenes, the highest ever reported in transgenic plants. However, largeheterologous proteins are not always processed correctly. Furthermore,such proteins are subject to attack by proteolytic enzymes afterformation. The cholera toxin B subunit is about 11 kilodaltons in size.The anthrax protective antigen is about 83 kd in size, presenting asignificant challenge to production in a chloroplast. A similarchallenge is presented by the F1-V antigen, which is a fusion protein.Such fusion proteins have not heretofore been successfully expressed inchloroplasts. Chloroplasts are prokaryotic in nature and express nativebacterial genes (like B subunit of cholera toxin) at very high levels(410-fold higher than nuclear expression). Production in chloroplasts isin sharp contrast to nuclear expression, that often requires extensivecodon modifications because of high AT content, unfavorable codons,presence of mRNA destabilizing sequences, and cryptic polyadenylation orsplice sites. Chloroplast transformation typically utilizes two flankingsequences that, through homologous recombination, insert transgenes intospacer regions between functional genes of the chloroplast genome, thustargeting transgenes to a known location. This eliminates the “positioneffect” and gene silencing frequently observed in nuclear transgenicplants. Chloroplast genetic engineering is an environmentally friendlyapproach, minimizing concerns of out-cross of introduced traits viapollen to weeds or other related crops. The chloroplast genome may beengineered without the use of antibiotic resistant genes as well for thedevelopment of edible vaccines. The term “edible vaccine” as used hereinrefers to a substance which may be given orally which will elicit aprotective immunogenic response in a mammal.

The difficulty of engineering the chloroplast genome without antibioticresistant genes has recently been overcome by modifying chloroplastswithout these genes. Engineering genetically modified crops withoutthese genes eliminates their potential transfer to the environment andto microbes in the gut. Antibiotic-free selection can be accomplished byusing the betaine aldehyde dehydrogenase (BADH) gene from spinach as aselectable marker. Specifically, the Applicant's published application,WO01/64023, which is hereby incorporated by reference, demonstratesusing an antibiotic free selectable marker. The selection processinvolves conversion of toxic betaine aldehyde (BA) to non-toxic glycinebetaine, which also serves as an osmoprotectant and helps confer droughttolerance.

The Applicant has transformed the chloroplast of edible monocot anddicot species, as is illustrated in the accompanying Figures.Specifically, the figures illustrate the stable transformation of carrotand corn chloroplast genomes, which has not previously beenaccomplished. The transformation of carrot, tomato, and corn allow theproduction of edible vaccines. These transformed plants also allow thepurification and use of the antigens produced by these plants as highpurity parenteral vaccines. However, the production of edible vaccinesis preferred for their ease of use and low cost.

FIGS. 17A and 18A illustrate the construction of maize chloroplasttransformation vector, where flanking regions were amplified using PCR.The PCR products were cloned and the expression cassette was inserted inthe transcriptionally active spacer region between trnI/trnA genes. Theexpression cassette of FIG. 17A has the Prrn promoter driving theexpression of GFP and BADH, which are regulated by (5′) gene10/rps16 3′and psbA 5′/3′ UTRs respectively. The expression cassette of 18A has thePrrn promoter driving the expression of aadA and BADH. The latter geneis regulated by (5′) gene10/rps16 3′ UTRs.

Functions of the genes in the carrot chloroplast transformation vectorswere tested in E. coli. For observing GFP expression, cells were platedon LB agar (Amp) plates and incubated at 37.degree. C. overnight. Cellsharboring pDD34-ZM-GFP-BADH were seen to fluoresce when exposed to UVlight, as is seen in FIG. 17B. To test the aadA gene expression, cellsharboring pDD33-ZM-aadA-BADH plasmid were plated on LB agar platescontaining spectinomycin (100 mg/ml) and incubated at 37.degree. C.overnight. Transformed cells grow on spectinomycin, as can be seen inFIG. 18B.

FIG. 19 shows GFP expression in embryogenic maize cultures studied underthe confocal microscope. FIG. 19A is a non-transgenic control, whileFIG. 19B-C are transformed maize embryogenic calli.

The selection in FIG. 19 was initiated two days after bombardment bytransferring the bombarded calli to callus induction medium containingBA or streptomycin. After eight weeks, a number of the healthy growingcalli from different bombardment experiments were examined for GFPexpression under the fluorescent stereomicroscope and the confocalmicroscope. Somatic embryos were regenerated on maize regenerationmedium containing BA or streptomycin.

FIG. 20A shows maize plants on regeneration medium containingstreptomycin or betaine aldehyde. FIG. 20A illustrates maize chloroplasttransgenic plants which were capable of growth on the selection agentindicating that construction of transgenic maize, while untransfomedmaize plants did not grow on the selection medium.

FIG. 20B shows PCR confirmation of chloroplast transgenic plants usingappropriate primers. Lanes 1-3, plants transformed withpDD34-ZM-gfp-BADH and Lanes 4-5, plants transformed withpDD33-ZM-aadA-BADH. Lanes − and + represent the negative and positivecontrols respectively. Genomic DNA was isolated from the leaf tissuesand PCR was performed on transformed and non-transformed tissues usingappropriate primers.

FIGS. 21A and 22A show the schematic construction of carrot chloroplasttransformation vectors. The construction of the carrot chloroplasttransformation vector illustrated in FIGS. 21A and 22A have flankingregions that were amplified using PCR. The PCR products were then clonedand the expression cassette was inserted in the transcriptionally activespacer region between trnI/trnA genes. The expression cassette of FIG.21A has the Prrn promoter driving the expression of GFP and BADH, whichare regulated by (5′) gene10/rps16 3′ and psbA 5′/3′ UTRs respectively.The expression cassette of FIG. 22A has the Prrn promoter driving theexpression of aadA and BADH. The latter gene is regulated by (5′)gene10/rps16 3′ UTRs.

Functions of the genes in the carrot chloroplast transformation vectorswere tested in E. coli. For observing GFP expression, cells were platedon LB agar (Amp) plates and incubated at 370 C overnight. Cellsharboring pDD37-DC-GFP-BADH were seen to fluoresce when exposed to UVlight (A). To test the aadA gene expression, cells harboringpDD36-DC-aadA-BADH plasmid were plated on LB agar plates containingspectinomycin (100 mg/ml) and incubated at 370 C overnight. Transformedcells grow on spectinomycin (B).

Functions of the genes in the carrot chloroplast transformation vectorswere tested in E. coli. For observing GFP expression, cells were platedon LB agar (Amp) plates and incubated at 37.degree. C. overnight. Cellsharboring pDD34-ZM-GFP-BADH were seen to fluoresce when exposed to UVlight as is seen in FIG. 21B. To test the aadA gene expression, cellsharboring pDD33-ZM-aadA-BADH plasmid were plated on LB agar platescontaining spectinomycin (100 .mu.g/ml) and incubated at 37.degree. C.overnight. Transformed cells grow on spectinomycin, as is seen in FIG.22B.

FIG. 23 (A-D) shows expression of GFP in different stages of transgeniccarrot cultures studied under confocal microscope (A) Untransformedcontrol, (B) Embryogenic callus, (C) Embryogenic callus differentiatedinto globular somatic embryos and (D) Somatic embryo with differentiatedcotyledons.

The aforementioned transformation of maize and carrot chloroplastprovides a novel approach for improved vaccines with the creation of anedible vaccine, which provides a heat stable environment, allows easyadministration at lower cost, and stimulates the mucosal and systemicimmune responses. There still remain a number of hurdles which need tobe overcome, such as the fact that protective antigens tend to be verylarge and unstable proteins (83 kDa); such large proteins have neverbeen expressed before in transgenic chloroplasts, and to date theApplicant is unaware of the expression and assembly of heptamers intransgenic chloroplasts.

Based upon the vector construct described above and set-forth in furtherdetail in this application, it is possible to have a general constructin edible plants where it can be determined through experimentationwhich construct can elicit the strongest immune response to bacterialtoxin challenges such as, but not limiting, plague or anthrax. As anexample, FIG. 16 offers a schematic of a construct as an example ofconstruct which could elicit an immune response to anthrax where, inthis non-limiting example, gene X is chosen from; 1) LF27-PA63; 2)CTB-LF27; 3) LF27-PA63+CTB-LF27; 4) LF27+PA. These antigens derived fromthese genes have been shown to elicit the immune response to anthrax. Itis noted however that a number of known genes, which code for abacterial antigen could be utilized in the construct.

Edible vaccines are heat stable unlike conventional vaccines which tendto be heat sensitive. An edible vaccine has a longer shelf life and doesnot require expensive refrigeration equipment. The heat sensitivity ofthe conventional vaccine makes it expensive, capricious and destinationlimited with a series of refrigeration steps during the transportationprocess from manufacture to final destination. Another major cause forthe high cost of biopharmaceutical production is purification; forexample, during insulin production, chromatography accounts for 30% ofthe production cost and 70% of the set up cost. Therefore, oral deliveryof properly folded and fully functional biopharmaceuticals couldpotentially reduce production costs by 90%. In one study it wasestimated that the banana could deliver a vaccine (Hepatitis B) at twocents per dose compared to approximately $125 per dose for conventionalHepatitis B vaccine injection. Bioencapsulation of pharmaceuticalproteins within plant cells offers protection against digestion in thestomach while allowing successful delivery. In three human clinicaltrials performed with plant derived vaccines, plant cells provedsufficient for vaccinogen protection against digestion, and theresulting vaccinogen induced systemic and mucosal immune responseswithout the aid of adjuvants. Oral administration of PA has also beenreported to offer protection against B. anthracis using an attenuatedSalmonella strain expressing PA. However, in all these studies thelimitation is the low levels of antigen expression which is overcome bythe present invention.

EXAMPLES Chloroplast Transformation

It should be understood that the examples set forth herein arenon-limiting examples, and the vectors can be used on a number ofplants. The following description is capable of being utilized asillustration and guide work for transformation of plants to express anybacterial antigen gene. It can also be used to express viral antigengenes.

Example 1 The pLD-JW1 Vector

FIG. 1 shows tobacco constructs and PCR confirmation of chloroplasttransgene integration. FIG. 1A shows the pLD-JW1 vector used forchloroplast transformation. The trnI and trnA genes were used asflanking sequences for homologous recombination. The constitutive 16srRNA promoter (“16s” in FIG. 1C) was used to regulate transcription. TheaadA gene conferring spectinomycin resistance was used for selection oftransgenic shoots. The pag gene coding for anthrax protective antigenwas regulated by the psbA promoter and 5′ (5UTR) and 3′ UTR (T)elements. As shown in FIG. 1B, The pLD-JW2 construct was made by addingorf1,2 from B. thuringiensis to the pLD-JW1 vector. FIG. 1C shows ascheme for PCR using the primers 3P and 3M to investigate chloroplasttransgene integration. The 3P primer anneals to the native chloroplastgenome and the 3M primer anneals to the aadA gene, generating a 1.65 kbPCR product in chloroplast transgenic lines. FIG. 1D shows the resultsof an analysis of randomly selected clones. Lane 1: Ladder; Lane 2:Negative control wild type tobacco plant DNA; Lane 3: Positivetransgenic plant DNA (pLD-5′UTR/HIS/THR/IFN.alpha.2b); Lane 4-8: 5different transgenic lines tested.

The pLD-JW1 vector (8.3 kb, see FIG. 1A) was constructed to transformtobacco chloroplasts. This construct is based on the vector pLD theApplicant has used successfully in previous publications. Specifically,the construct is connected to the Applicants universal vectors, whichare described in detail in PCT patent publication WO 99/10513, which ishereby incorporated by reference. The trnI and trnA genes were used asflanking sequences for homologous recombination to insert apag-containing cassette into the spacer region between these two tRNAgenes in the inverted repeat region of the chloroplast genome, asreported previously. It should be noted that it is possible to insertthe pag containing cassette into any of a number of spacer regionsbetween genes. The constitutive 16s rRNA promoter, which can berecognized by both the chloroplast encoded RNA polymerase and thenuclear encoded RNA polymerase, was used to drive transcription. Any ofa number of promoters functional in plastids and well understood andknown in the art can be used to help drive transcription.

The aadA gene conferring spectinomycin resistance was used for selectionof transgenic shoots. It is noted however, that the use otherantibiotic-resistant genes or an antibiotic free selectable marker suchas BADH, could also be used in the construction of the vector. The paggene coding for anthrax protective antigen was regulated by the psbA 5′and 3′ elements. The 5′UTR from psbA, including its promoter, was usedfor transcription and translation enhancement and the 3′UTR regionconferred transcript stability.

One skilled in the art would recognize that an alternative chloroplastvector can be constructed without the use of any promoter, because allpolycistronic spacer regions contain a native promoter which can be usedto drive transcription.

A second construct was made by adding orf1,2 from B. thuringiensis tothe pLD-JW1 vector, forming the pLD-JW2 vector (see FIG. 1B). The orf1,2gene codes for a putative chaperone. Including the orf1,2 gene was doneto test whether the putative chaperone could fold a heterologousBacillus protein (i.e., PA) into cuboidal crystals or form inclusionbodies, protect PA from proteolytic degradation, and thereby facilitatepurification.

Transgene Integration into the Chloroplast Genome by PCR Analysis:

Chloroplast transgenic lines were generated by particle bombardment asdescribed previously. After bombarding Nicotiana tabacum cv. PetitHavana tobacco leaves with the chloroplast vectors, the leaves weregrown on selective medium containing 500 .mu.g/ml spectinomycin. Threedifferent genetic events can produce spectinomycin-resistant tobaccoshoots: (1) transgene integration into chloroplasts, (2) into thenuclear genome or (3) spontaneous mutants. PCR with two specificprimers, 3P and 3M, allowed identification of shoots having the desiredchloroplast transgene integration. The 3P primer annealed to the nativechloroplast genome and the 3M primer annealed to the aadA gene (see FIG.1C). Nuclear transformants were eliminated because 3P will not annealand mutants were ruled out because 3M will not anneal. At least 50shoots were obtained in the initial transformation, a high frequencywhich suggests that there was no inhibitory effects of PA. Among clonestested, in both constructs, all were chloroplast transformants. Nospontaneous mutants or nuclear transformants were observed (see FIG.1D).

Chloroplast Integration of Transgenes and Homoplasmy:

Southern blots were done to further verify that the transgenes had beenintegrated into the chloroplast genome and to determine homoplasmy(containing only transformed chloroplast genomes) or heteroplasmy(containing both transformed and untransformed chloroplast genomes).Total plant DNA was digested with the enzyme BglII which generated a4.4-kb fragment in wild type, 5.2-kb and 3-kb fragments in pLD-JW1transgenic lines, and 6.8-kb and 3-kb fragments in pLD-JW2 transgeniclines when hybridized with a 0.81-kb probe made from chloroplastflanking sequences (see FIG. 2A-D). The blots were also hybridized witha 0.52-kb probe made from pag coding sequence (see FIG. 2E). All of thepLD-JW2 plants appeared to be homoplasmic. In the pLD-JW1 plants,transgenic line 2 appeared to be heteroplasmic, which is not uncommon tofind in T.sub.0 plants. T.sub.0 refers to first generation transgeniclines and T.sub.1 refers to second generation lines obtained bygermination of seeds from T.sub.0.

FIG. 2 shows southern blots to investigate the site of transgeneintegration and determine homoplasmy or heteroplasmy. The figure shows aschematic diagram of the expected products from digestion of wild typeuntransformed plant as follows: FIG. 2A shows plants transformed withpLD-JW1; FIG. 2B shows plants transformed with pLD-JW2. FIG. 2D shows aflanking sequence probe revealing heteroplasmy in pLD-JW1 line andhomoplasmy in pLD-JW2 lines. FIG. 2E shows pag sequence probe showingpresence of pag in transgenic lines.

PA Expression in Transgenic Chloroplasts:

Western blots were performed on transgenic lines containing the twodifferent constructs. Full length 83-kDa polypeptide was detected/onblots, confirming PA expression in transgenic lines and absence ofunique proteases that cleave PA in plant cells (see FIGS. 3A-C).Presence of active furin or trypsin-like proteases would have resultedin a 63-kDa protein due to cleavage at the sequence RKKR (amino acids164-167). The sequence FFD at residues 312-314 is another site that ishighly sensitive to chymotrypsin-like enzymes, and cleavage would haveresulted in 47- and 37-kDa fragments. No other cleaved PA products wereobserved (see full length blots shown), demonstrating stability ofchloroplast derived PA. Prior to the Applicant's discovery, it waswidely believed that proteases would cleave long antigenic bacterialpeptides, but this invention illustrates that the antigenic bacterialpeptides are free from protolytic degradation.

The T.sub.1 transgenic lines showed a greater amount of PA in thepLD-JW1 lines as compared to the pLD-JW2 lines. Additional western blotswere done comparing two different detergents in the extraction buffers,CHAPS and SDS, and both were found to extract PA equally well (see FIG.3C). The supernatant and the homogenate were also found to be comparablesuggesting that most of the PA is in the soluble fraction. After storagefor two days at 4.degree. C. and −20.degree. C., the PA in plant crudeextracts is quite stable (see FIG. 3C). Powdered leaf was stored at−80.degree. C. for several months before performing western blots orfunctional assays; this did not result in any noticeable decrease in PAquantity or functionality. This facilitates long term storage ofharvested leaves before extraction of PA for vaccine production. NativePA has been shown to be highly unstable due to proteolysis-sensitivesites, which have been modified to confer better stability.

Quantification of PA in Transgenic Chloroplasts:

In order to quantify the amount of PA in transgenic chloroplasts,western blots were used to observe varying dilutions of the crudeextract. PA was quantified using gel documentation software (Bio-Rad).Based on western blot analysis, pLD-JW1 T.sub.1 transgenic lines showed44 .mu.g PA/g of fresh tissue (22 .mu.g/ml). An average tobacco leafweighed 6.5 g; therefore 286 .mu.g PA could be expressed per leaf. ThepLD-JW2 transgenic lines showed lower levels of PA accumulation,probably due to interference of both UTRs, resulting in decrease intranslation. We have observed recently that the combination ofORF-psbA5′UTR decreases expression of human serum albumin in transgenicchloroplasts.

The psbA regulatory sequences, including the promoters and UTRs, havebeen shown to enhance translation and accumulation of foreign proteinsunder continuous light. Therefore, pLD-JW1 T.sub.1 transgenic plantswere placed in continuous light and young, mature, and old leaves werecollected after 3 or 5 days of continuous illumination. Western blotswere performed using different dilutions of crude plant extracts (seeFIG. 4A-B). The 3 day mature and young leaves contained 80 .mu.g PA or108 .mu.g PA/g of fresh tissue. The 5 day old, mature and young leavescontained 32 .mu.g PA, 108 .mu.g PA or 156 .mu.g PA/g of fresh tissue,respectively. Thus, young leaves showed the highest accumulation of PAand old leaves showed the lowest, probably due to proteolyticdegradation induced during senescence. These assays quantified only fulllength PA. In spite of loading 500-1000 fold more protein ofuntransformed plant extracts, no cross-reacting protein was observedwith the monoclonal antibody used.

FIG. 4 shows total protein from plant extracts loaded in each lane isshown in parenthesis. FIG. 4A shows pLD-JW1 transgenic line in 3 days ofcontinuous light, 1:20 dilutions. Lane 1: old leaf (187 ng); Lane 2:mature leaf (369 ng); Lane 3: young leaf (594 ng); Lanes 4-5: blank;Lane 6: 10 ng PA; Lane 7: 20 ng PA; Lane 9: ladder; Lane 10: wild type(15,000 ng). FIG. 4B shows pLD-JW1 transgenic line in 5 days ofcontinuous light, 1:20 dilutions. Lane 1: old leaf (214 ng); Lane 2:mature leaf (588 ng); Lane 3: young leaf (745 ng); Lane 6: 10 ng PA;Lane 7: 20 ng PA; Lane 8: blank; Lane 9: ladder; Lane 10: wild type(15,000 ng). FIG. 4C is a histogram of .mu.g PA/g fresh tissue in young,mature, and old leaves after 3 (blue) and 5 (red) days of continuousillumination.

PA accumulation was visualized in crude plant extracts by Coomassiestaining. When a capture ELISA was used to quantify PA, it appeared thatPA constituted a large percentage of total soluble protein in someextracts. While these values may reflect detection of partially cleavedPA, they do not result from non-specific interaction of the antibodieswith any other proteins, because no signal was detected in untransformedplants. However, the data set forth herein is from quantitative scanningof polyacrylamide gels and not from the capture ELISA.

PA Functionality Determined by Macrophage Lysis:

Supernatant and homogenate samples from both To constructs, pLD-JW1 andpLD-JW2, were tested. Two different buffers were used to extractproteins—one contained CHAPS detergent and one did not have anydetergent. The PA produced in chloroplast transgenic lines was able tobind to the anthrax toxin receptor, be cleaved to the 63-kDa fragment,heptamerize, bind LF, be internalized and lyse the macrophage cells.Therefore the transgenic plants were shown to produce fully functionalPA (see FIG. 5A). Active PA was found in both the supernatant andhomogenate fractions; but was quantitated only in the former. The assayusing CHAPS gave a result of the pLD-JW1 supernatant with the bestyield. In the absence of any detergent, the supernatants lysed themacrophage cells better than the homogenates. Crude plant extractscontained up to 5 .mu.g per ml functional PA confirming expression ofhigh levels of functional PA. Supernatant samples from T1 pLD-JW1transgenic lines were tested and they resulted in approximately 12-25.mu.g functional PA per ml (see FIG. 5B) and the toxicity was entirelydependent on the presence of LF.

The threat of biological warfare and terrorism is real. The mosteffective way to prevent or deter effective use of anthrax as a weaponwould be to produce an efficacious and inexpensive vaccine. Plants arean inexpensive and easy way to produce recombinant proteins, withouthuman or animal pathogen contamination. Because one acre of tobaccoyields up to 40 tons of fresh leaves (40,000 kg in three cuttings), theproduction could be up to 6.24 kg PA per acre based on expression levelsreported in this manuscript. There is less than 50% loss duringpurification from plant extracts (loss of foreign protein is generallybetween 30 and 90%), and at 5 .mu.g PA per dose (which is roughlyequivalent to prior art vaccine which is in a range of 1.75 to 7 .mu.gPA), one can produce 600 million doses of vaccine per acre of tobacco.Thus a few acres of transgenic tobacco can meet the world's need for theanthrax vaccine.

Experiment Protocol for Example 1 Bombardment and Selection ofTransgenic Plants

Sterile Nicotiana tabacum cv. Petit Havana tobacco leaves were bombardedusing the Bio-Rad PDS-1000/He biolistic device as previously described.The bombarded leaves were placed on RMOP medium containing 500 .mu.g/mlspectinomycin for two rounds of selection on plates and subsequentlymoved to jars of MSO medium containing 500 .mu.g/ml spectinomycin.

PCR Analysis to Test Stable Integration:

DNA was extracted from tobacco leaves using Qiagen DNeasy Plant Mini Kitavailable from Qiagen, Valencia, Calif. PCR was performed using thePerkin Elmer Gene Amp PCR System 2400 (available from Perkin Elmer,Chicago, Ill.). PCR reactions contained template DNA, 1.times.Taqbuffer, 0.5 mM dNTPs, 0.2 mM 3P primer, 0.2 mM 3M primer, 0.05units/.mu.l Taq Polymerase, and 0.5 mM MgCl.sub.2. Samples were run for30 cycles as follows: 95.degree. C. for 1 min, 65.degree. C. for 1 min,and 72.degree. C. for 2 min with a 5 min ramp up at 95.degree. C. and a72.degree. C. hold for 10 min after cycles complete. PCR products wereseparated on 1% agarose gels.

Southern Blot Analysis:

Total plant DNA was digested with BglII and run on a 0.8% agarose gel at50 V for 2 hours. The gel was soaked in 0.25 N HCl for 15 minutes andthen rinsed 2.times. with water. The gel was soaked in transfer buffer(0.4 N NaOH, 1 M NaCl) for 20 minutes and then transferred overnight toa nitrocellulose membrane. The membrane was rinsed twice in 2.times.SSC(0.3 M NaCl, 0.03 M Sodium citrate), dried on filter paper, and thencrosslinked in the GS GeneLinker (Stratagene, La Jolla, Calif.). Theflanking sequence probe was made by digesting pUC-CT vector DNA withBamHI and BglII to generate a 0.81 kb probe. Lee, S. B., Byun, M. O.,Daniell, H. Accumulation of trehalose within transgenic chloroplastsconfers drought tolerance. Molecular Breeding (in press) (2002). The pagprobe was made by digesting pag with NcoI to generate a 0.52 kb probe.The probes were labeled with .sup.32P using the ProbeQuant G-50 MicroColumns (Amersham, Arlington Heights, Ill.). The probes were hybridizedwith the membranes using Stratagene QUICK-HYB hybridization solution andprotocol (Stratagene, La Jolla, Calif.).

Western Blot Analysis:

Approximately 100 mg of leaf tissue was ground in liquid nitrogen with amortar and pestle and stored at −80.degree. C. for several months. Whenit was time to extract the proteins, the powder was removed from−80.degree. C. and 200 .mu.l of plant extraction buffer was added andmixed with mechanical pestle (0.1% SDS, 100 mM NaCl, 200 mM Tris-HCl pH8.0, 0.05% Tween 20, 400 mM sucrose, 2 mM PMSF). The plant extract wasthen centrifuged for 5 minutes at 10,000.times.g to pellet the plantmaterial. The supernatant containing the extracted protein wastransferred to a fresh tube and an aliquot was taken out, combined withsample loading buffer, boiled, and then run on 8% SDS-PAGE gels for onehour at 80 V, then 2 hours at 150 V. Gels were transferred overnight at10 V to nitrocellulose membrane. The membrane was blocked with PTM(1.times.PBS, 0.05% Tween 20, and 3% dry milk). PA was detected withanti-PA monoclonal antibody 14B7. Secondary antibody used was goatanti-mouse IgG conjugated to horseradish peroxidase (American QualexAntibodies, A106PN).

The stability assay utilized SDS buffer (0.1% SDS, 100 mM NaCl, 10 mMEDTA, 200 mM Tris-HCl pH 8.0, 0.05% Tween 20, 14 mM.beta.-mercaptoethanol, 400 mM sucrose, 2 mM PMSF) and CHAPS buffer (4%CHAPS, 100 mM NaCl, 10 mM EDTA, 200 mM Tris-HCl pH 8.0, 14 mM.beta.-mercaptoethanol, 400 mM sucrose, 2 mM PMSF). Two hundred .mu.l ofeach buffer was added to 100 mg powdered leaf tissue. For supernatantfractions, the extraction was centrifuged at 10,000.times.g for 5minutes and supernatant was removed. For homogenate, the entire extractwas used. The samples were stored at 4.degree. C. and −20.degree. C. fortwo days. The rest of the western protocol was the same as describedabove. Dilutions of 1:10 and 1:20 of the protein extracts were made andrun on the gel along with 20, 10, and 5 ng of PA protein standards togenerate a standard curve for protein quantification. After the film wasdeveloped, the PA was quantified using the Gel-Doc.

Macrophage Lysis Assays:

Approximately 100 mg of powdered leaf tissue was extracted with 200.mu.l of extraction buffer. For the supernatant fraction, the buffer andtissue were centrifuged for 5 minutes at 10,000.times.g and thesupernatant was placed in a new tube. For the homogenate, it was all ofthe tissue and the buffer. RAW 264.7 macrophage cells were plated in96-well plates in 120 .mu.l DMEM medium and grown to 50% confluence. Themedium was aspirated and replaced with 100 .mu.l medium containing 250ng/ml LF. The control plate received medium with no LF to test toxicityof plant material and buffers. In separate 96-well plates, the plantsamples were diluted serially 2-fold and 40 .mu.l of the dilutions weretransferred onto the RAW cells so the top row had plant extract at 1:14dilution. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) was added after 5-10 hours to assess cell death. Gu, M. L.,Leppla, S. H., Klinman, D. M. Protection against anthrax toxin byvaccination with a DNA plasmid encoding anthrax protective antigen(Vaccine. 17, 340-344 (1999)).

Example 2 Transformation of the Tomato Chloroplast Genome

Prior to the creation of the pTom-BADH2-G10-pag ˜8.8 kb vector construct(FIG. 7), the Applicant constructed the pLD Tom-BADH vector (FIG. 6)illustrating the ability to transform the chloroplast genome of edibledicots. The Tom-BADH vector was constructed to with two selectablemarker genes (BA and spectinomycin) to test ability of transformedplants to grow on BA as compared to spectinomycin. The pLD-Tom-BADHvector contains the chimeric aadA gene and the BADH gene driven by theconstitutive 16 S rRNA promoter and regulated by the 3′UTR region ofpsbA gene from petunia plastid genome. In this construct both, aadA andBADH possess the chloroplast preferred ribosomal binding site, GGAGG.Another suitable vector used for tomato chloroplast transformation isthe pLD-Tom-UTR-BADH vector, which has the constitutive 16 S rRNApromoter driving the expression of the dicistron, in which the BADH isunder the regulation of the promoter and the 5′UTR of the psbA gene andthe 3′ UTR of psbA gene, for enhanced expression.

After successful construction and integration into the tomatochloroplast genome using the pLD Tom-BADH vector of FIG. 6, theApplicant then constructed the pTom-BADH2-G10-pag ˜8.8 kb vector, whichis illustrated in FIG. 7. FIG. 7 shows the schematic construct of thepTom-BADH2-G10-pag ˜8.8 kb vector, which was constructed containing theselectable marker gene BADH. This construct is also be made using theaad gene to confer spectinomycin resistance in place of the BADH gene.After bombarding the tomato cotyledons (seed leaves or embryonic leaves)with the tomato construct of FIG. 7, the cotyledons were put onselection media containing Betaine Aldeyhyde (BA) and calli formed. Thecalli were transferred to new selection media to obtain shoots.

FIG. 11 shows the first selection after bombardment with thepTom-BADH2-G10-pag ˜8.8 kb vector, wherein the cotyledons were incubatedin the dark for 48 hours and then the bombarded cotyledons were cut. TheRMOP medium shown in FIG. 11 is a shoot inducing media. The bombardedcotyledons were grown on 2.5 mM Betaine Aldehyde (BA) for selection.

FIG. 8 shows the PCR test that was performed to determine integration ofthe pTom-BADH2-G10-pag ˜8.8 kb vector, where the tomato shoots weretested with PCR to confirm integration of the transgene. In FIG. 8,the + is pTOM-G10-PA vector control, − is WT Tomato plant, and #3 is thetransgenic tomato plant. This confirmation utilized appropriate primers.

To apply the plastid transformation technology to edible plants toproduce an edible vaccine, a first generation tomato vector isconstructed containing pag. TrnI and trnA are homologous recombinationregions in tomato; 5′UTR from psbA is used for translation enhancementand also contains it's own promoter; BADH gene confers Betaine Aldehyde(BA) resistance; G10 is a translation enhancer from the T7bacteriophage; pag codes for the protective antigen, and T is the psbAterminator.

The pDD11 vector is cleaved with NdeI & NotI to remove the gene andleave opened pBlue-G10 region of the vector. The pBlue-T7-pag vector isthen cleaved with NdeI & NotI to remove the pag gene .about.2.2 kb. Thepag gene is ligated into the opened pBlue-G10 vector, and the resultingpBlue-G10-pag is cleaved with SmaI & NotI to remove the G10-pag.about.5.2 kb segment from the pBlue-G10-pag vector. Finally thepTom-BADH2 vector is cleaved with SmaI and NotI, and then the G10-pagfragment is ligated into the vector creating a pTOM-BADH2-G10-pag ˜8.8kb (FIG. 7).

PCR Confirmation of Transgene Integration:

After bombarding the tomato cotyledons (seed leaves or embryonic leaves)with the tomato construct vector, pTOM-BADH2-G10-pag ˜8.8 kb, thecotyledons are put on selection media containing Betaine Aldeyhyde (BA)and calli formed (FIG. 11). The calli are transferred to new selectionmedia to obtain shoots. Shoots are tested with PCR to confirmintegration of the transgene, which utilizes appropriate primers (FIG.8).

Mucosal and Transcutaneous Immunization Against Anthrax:

Anthrax vaccine studies focused on mucosal and transcutaneousimmunization with rPA as a vaccine antigen when delivered in conjunctionwith a novel adjuvant, designated LT(R192G). This adjuvant was shown tobe effective at augmenting protection against a variety of bacterial,viral, and fungal pathogens when delivered with appropriate antigensintranasally, orally, rectally, or transcutaneously. This adjuvant wasdeveloped by the Clements laboratory at Tulane University HealthSciences Center with funding from NIH and the Department of Defense andhas been evaluated in a number of Phase I and Phase II clinical trials.In rPA studies, a number of immunologic outcomes were measured, but thestudies focused primarily on those associated with protection againstinhaled pathogens—serum and bronchial lavage (BAL) fluid antibodies. Itwas demonstrated that mucosal and transcutaneous immunization of micewith rPA induces high levels of antigen-specific antibodies in serum andin bronchoalveolar lavage fluids. Moreover, circulating anti-rPAantibodies are able to neutralize the cytotoxic effect of anthrax LethalToxin when tested in macrophage cytotoxicity assays. FIG. 12 shows theserum anti-PA as determined by ELISA and the in vitro toxinneutralization by serum antibodies following intranasal immunization.Equivalent results were seen following transcutaneous immunization.

Tomato Chloroplast Integration Vector:

Since the expression of the foreign protein is desired in chromoplastsof tomato fruit, the gene of interest needs to be under the control of aregulatory sequence that is free from cellular control. In this context,examples of suitable candidate regulatory sequences are the T7 gene 10leader sequence and cry2Aa2 UTR. The T7 gene 10 leader sequence is usedto express foreign proteins in transgenic chromoplasts. The cry2Aa2 UTRaccumulates foreign protein in chromoplasts as efficiently as the psbAUTR. The selectable marker for the future generation vectors canoptionally be the BADH gene under the regulation of psbA promoter and5′UTR as psbA is one of the most efficiently translated chloroplastgenes in green tissues. Since green tissue is used for introducing thetransgene into the chloroplasts in tomato, it is ideal to use the lightregulated psbA UTR for the selectable marker.

Tomato Seed Sterilization and Growth Conditions:

FIG. 9 shows tomato seeds (Moneymaker and Ady varieties) that aresurface sterilized with ethanol for 30 s, followed by a 20 min treatmentwith 1.5% NaOCl and 0.1% Tween 20. Seeds are washed thoroughly withsterile water (at least 3-4 times) and transferred to germination media(FIG. 14). Germination media consists of MS salts with 30% sucrose and0.8% agar. About 20 seeds are inoculated per bottle and placed under aphotoperiod of 16 h light and 8 h dark for 8-10 days to obtaincotyledons for particle bombardment. The cotyledons are then excisedeither as an explant for bombardment or the resulting seedlings are usedfor transplantation to obtain leaves.

The cotyledons and leaf material are bombarded using the particle gun.After bombardment the explants are then incubated in the dark for 48 h.The cotyledons and leaves are then cut into small pieces and placed ontoRMOP media supplemented with 2.5, 5.0 and 7.5 mM of betaine aldehyde forregeneration. In the case of cotyledons, the concentration of 2.5 nm BAis optimal, but not required, as regeneration of putative transformantscould be observed after two weeks. Specifically, there is no response onmedia having higher concentrations of 5.0 and 7.5 mM BA. With leaves,the concentration of 1 and 1.5 mM is optimal for pLD-Tom-BADH andpLD-Tom-UTR-BADH respectively.

Preparation and Analysis of Stable Tomato Plant Transformants:

Selection is optimally performed in the presence of BA, but has alsobeen performed in the presence of antibiotics. After selection, PCRanalysis is performed as described above, as is well understood in theart. Finally Southern and northern blot analyses are performed asdescribed above, and well understood in the art, to determine the amountand level of transformation in the chloroplast genome.

Example 3 Transformation of Carrot to Produce Anthrax Vaccine

Carrot (Daucus carota L.) is a biennial plant grown for its edibletaproot. It is one of the most important vegetables used worldwide forhuman consumption. Carrot taproots are rich in vitamin A and fiber andare ideal to genetically manipulate in the chromoplast for theproduction of edible vaccines. For transformation of carrot, flankingsequences (trnI and trnA) are amplified with the help of PCR. Durationfor regeneration of carrot plantlets is shortened to four months fromeight months when replacing the antibiotic selection with BA. The samechloroplast constructs as described above for tomatoes are used forcarrot except that homologous recombination regions i.e. trnI and trnAare derived from carrot chloroplast DNA. The advantage of using carrotis that from small clusters of cells or a small piece of carrot one canget thousands of transgenic plants in a limited space. Moreover, singlecells are directly in contact with the culture media surface. Therefore,even a small quantity of selecting agent (betaine aldehyde) is moreeffective in comparison to other larger tissues. Carrot is easy to storefor long periods of time.

Plant Material and Tissue Culture:

Seeds of carrot (Daucus carota L. cv Nantaise) are sown in pots andplaced under a growth chamber with appropriate growth conditions for aslittle as four weeks to as long as a year. The hypocotyls are then cutinto segments of 1 cm long and placed either on semi-solid callusinduction medium or in 50 ml MS medium containing 3% sucrose, 0.1 mg/l2,4-dichlorophenoxyacetic acid (2,4-D) having pH 5.7. After 3 weeks ofcontinuous shaking at 24.degree. C. and 120 rpm, liberated cells arecollected on a 100 .mu.M mesh, centrifuged (150.times.g for lo min) andresuspended in fresh medium. Rapidly growing cell cultures can besubcultured weekly. Next, callus formation from hypocotyls segments isestablished on semi-solid MS medium (Carolina Biological supply company)containing 1 mg/l kinetin and 3 mg/l 2,4-D. Homogenously growing calliis subcultured every 4 weeks on fresh medium. The resulting friablecalli is then resuspended in 50 ml MS medium containing 3% sucrose and0.1 mg/kinetin. Finally, suspension-cultured cells are filtered througha 100 .mu.M mesh and subjected to bombardment with chloroplast vectors.

Bombardment and Regeneration of Carrot Chromoplast Transgenic Plants:

Fine cell suspension culture of carrot, evenly spread over MS semi-solidmedium is used for bombardment. After bombardment the explants areincubated in the dark for 48 h and later in appropriate light condition(16/8 h day/night cycle at 24.degree. C.). Somatic embryogenesis isinduced in a suspension of single cells and small clusters harvested onsieve and low-speed centrifugation. The harvested cells are washed oncewith hormone free liquid MS medium and resuspended in 40 ml hormone freeMS medium containing different concentrations of betaine aldehyde (1.5,2.5 and 3.5 mM). Transgenic somatic embryos, visible 2 weeks afterinduction, are selected manually and transferred onto plates withsemi-solid MS medium containing 1.5% sucrose and variable concentrationsof betaine aldehyde (1.5, 2.5 and 3.5 mM). The plates are sealed withparafilm. After two weeks, somatic embryos development into plantletswhich are transferred to soil in pots. Initially, the pots are coveredwith plastic bags to maintain high humidity and irrigated withprogressively reduced concentrations of MS salts for the first week,followed by tap water in the second week. Transgenic plants with stableexpression of recombinant protein are then utilized for suitable assays.

Example 4 Construction for the F1V Fusion Protein (the Plague Antigen)

The production of Yersinia pestis vaccine in a low nicotine strain oftobacco (LAMD) is accomplished by expressing in chloroplasts the F1-Vantigen fusion protein produced from F1 gene (513 bp/15.5 kDa) and theentire V antigen (980 bp/35 kDa). The entire immunogenic sequence willbe (441+980+6 for a hinge=1437 bp). With the protein of 478 amino acidshaving a calculated mass of 53,193 and a pI of 5.1 has shown this fusionprotein to be immunoprotective.

F1-V was modified to add an EcoR1 site. This fragment is cloned into theuniversal chloroplast vector, which has been described above, with thepsbA 5′UTR upstream of the F1-V fusion. The use of the psbA 5′UTR, isnot required, but it has been shown to increase expression of foreignproteins by chloroplast.

Large-scale expression of the fusion protein results in the formation ofinclusion bodies as observed with several other foreign proteinsexpressed in transgenic chloroplasts. These inclusion bodies are easilyseparated by centrifugation. Another option is use of ammonium sulphatefor the precipitation of the protein.

Optionally, a His-tag with an enterokinase cut site was added to theabove construct. The His-tag allows for purification on a nickel columnwith subsequent cleavage of the fusion protein from the His-tag.

The plasmid pPW731 (a pET-24 vector) carrying the gene for the F1Vfusion protein was delivered in BLR strain of E. coli. Because of theexonuclease activity in BLR, XL1-Blue strain of E. coli was transformedwith pPW731. Using NdeI and NotI, F1V was cut out of pPW731 and ligatedinto PCR2.1 with 5′ psbA. In order to ligate 5′psbA-F1V into theuniversal chloroplast vector, pLD-CtV, PCR2.1-5′ psbA-F1V was cut withSac I, and blunt ended, then cut with Not I. This was ligated intopLD-CtV which had been cut with EcoRV (blunt end) and Not I. Thisproduced the chloroplast vector pLDS-F1V containing the 5′UTR psbAupstream of F1V, which was then used for bombardment. The use of thepsbA 5′UTR has proven to increase expression of foreign proteins bychloroplasts.

FIG. 13A shows the construct of the pLDS-F1V vector, wherein the vectorcontains the F1/V antigen gene contained in the spacer region betweenthe trnI and trnA genes. It should be understood that the F1/V antigengene could be inserted into any of a number of spacer regions betweenchloroplast genes, which are described and illustrated in Sugita, M.Sugiura, M. Regulation of gene expression in chloroplast of higherplants, Plant Molecular Biology 32:315-326, 1996).

FIG. 13B shows PCR restriction enzyme analysis of pLDS-F1V with Xho I,Eco RI, and Nde I, which showed that the psbA-F1V sequence to be inproper orientation in pLD. (Xho I yielding: 340 bp, 2679 bp, and 4634bp: Eco RI yielding 682 bp, and 6953 bp: and Xho I/Nde I yielding 340bp, 925 bp, 1102 bp, 1620 bp, 3610 bp, and incomplete digestion at 2601bp, and 4550 bp). Lane 1:1 KB Ladder, Lane 2: pLDS 37 C, Lane 3: pLDS 4C, Lane 4: Eco RI, Lane 5: Xho I, Lane 6: Xho I/Nde I (overnight), Lane7: Xho I/Nde I. The sequencing of pLDS-(5′ psbA)-F1V using the 5′UTRprimer, which lands on the 5′ psbA, showed no changes during vectorconstruction.

FIGS. 14A and 14B show PCR confirmation of transgene integration intothe chloroplast genome.

After bombarding tobacco leaves with pLDS-F1V, there are threepossibilities that might produce shoots: chloroplast transgenic, nucleartransgenic, and mutants resistant to spectinomycin. In order to selectchloroplast transgenic plants we utilize two PCR reactions. The first(FIG. 14A), which checks for chloroplast intergration, uses 3P and 3Mprimers which land on the native chloroplast geneome and the aadA gene,respectively. Nuclear transformants are screened out because 3P will notanneal. Mutants are screened out because 3M will not anneal. Positivechloroplast transformants produce a 1.65 Kb PCR product.

The second PCR reaction (FIG. 14B) uses 5P which lands on the aadA geneand 2M which lands on trnA. This produces a PCR product of 1.65kb+Insert (psbA=203 bp+F1V=1437 bp)=3.29 Kb. Plants 2-5, 8, and 10clearly contain the transgene.

FIG. 15A shows the western blot of pLDS-F1V from XL1-Blue strain of E.coli, and 15B shows the western blot of plants 1 and 2.

Turning to FIG. 15A showing the Western blot of F1V expression in E.coli, the expression in E. coli was detected by rabbit anti-F1 as theprimary antibody and alkaline phosphatase labeled goat anti-rabbit IgGas the Western blot of F1V expression in E. coli was detected by rabbitanti-F1 as the primary antibody and alkaline phosphatase labeled goatanti-rabbit IgG as the secondary antibody. Specifically the western blotin FIG. 15A shows: Lane 1: pLDS-F1V; Lane 2: F1 antigen; Lane 3: Vantigen; Lane 4: F1V fusion protein.

FIG. 15B illustrates the Western blot of F1V expression in plants as wasdetected by rabbit anti-F1 and anti-V as primary antibodies and alkalinephosphatase labeled goat anti-rabbit IgG as the secondary antibody.Controls and samples were boiled. Specifically the western blot in FIG.15B shows: Lane 1: purified F1V fusion protein; Lane 2: UntransformedPetit Havana; Lane 3: Transformed plant line #1; Lane 4: Transformedplant line #2. From this western blot it is clear that transgenic line 2has surpassed line 1 in growth and is very healthy confirming that theforeign protein is not toxic to plants.

Cloning F1-V Antigen into Tomato:

The F1-V antigen, which is a bacterial antigen, was cloned into thetomato pLD vector between gene 10 and rps 16 terminator. This wasdiscussed further above. In this case, the selectable marker, BADH, withpsbA 5′ UTR and psbA 3′ follows the rps16 region. The second vector madecontains F1-V attached to the carboxy terminus of CTB. CTB serves as amucosal carrier for this plague protein.

Example 5 Oral Delivery of Recombinant Proteins Via Cholera Toxin BSubunit (CTB)

The increased production of an efficient transmucosal carrier moleculeand delivery system in chloroplasts of plants allows the production ofplant based oral vaccines and fusion proteins with CTB. CTB haspreviously been expressed in nuclear transgenic plants at levels of 0.01(leaves) to 0.3% (tubers) of the total soluble protein. To increaseexpression levels, the chloroplast genome was engineered to express theCTB gene. We observed expression of oligomeric CTB at levels of 4-5% oftotal soluble plant protein. PCR and Southern Blot analyses confirmedstable integration of the CTB gene into the chloroplast genome. Westernblot analysis showed that transgenic chloroplast expressed CTB wasantigenically identical to commercially available purified CTB antigen.Also, GM1-ganglioside binding assays confirm that chloroplastsynthesized CTB binds to the intestinal membrane receptor of choleratoxin Transgenic tobacco plants were morphologically indistinguishablefrom untransformed plants and the introduced gene was found to be stablyinherited in the subsequent generation as confirmed by PCR and Southernblot analyses. Thus chloroplasts form disulfide bridges to assembleforeign proteins. Spontaneously forming CTB pentamers exhibit intacttranscytosis to the external basolateral membrane of intestinalepithelium, and have been widely used as oral vaccine vehicles.

Example 6 Double Barrel Plastid Vectors

Chloroplast transformation has been accomplished only in a fewSolanaceous crops so far. There are several challenges in extending thistechnology to other crops. So far, only green chloroplasts have beentransformed in which the leaf has been used as the explant. However, formany crops, including monocots, cultured non-green cells or othernon-green plant parts are used as explants. These non-green tissuescontain proplastids instead of chloroplasts, in which gene expressionand gene regulation systems are quite different. During transformation,transformed proplastids should develop into mature chloroplasts andtransformed cells should survive the selection process during all stagesof development. Therefore, the major challenge is to providechloroplasts the ability to survive selection in the light and the dark,at different developmental stages. This is absolutely critical becauseonly one or two chloroplasts are transformed in a plant cell and theseplastids should have the ability to survive the selection pressure,multiply and establish themselves while all other untransformed plastidsare eliminated in the selection process. The Double Barrel PlastidVectors accomplish this by using genes coding for two different enzymescapable of detoxifying the same selectable marker (or spectrum ofselectable markers), driven by regulatory signals that are functional inproplastids as well as in mature chloroplasts.

The plastid vector described here is one among several such examples(non-limiting example). The chloroplast flanking sequence containsappropriate coding sequences and a spacer region into which thetransgene cassette is inserted. Any spacer sequence within the plastidgenome could be targeted for transgene integration, includingtranscribed and transcriptionally silent spacer regions. Both aphA-6 andaphA-2 (nptII) genes code for enzymes that belong to the aminoglycosidephosphotransferase family but they originate from different prokaryoticorganisms. Because of prokaryotic nature of the chloroplast genome,these genes are ideal for use in transgenic chloroplasts without anycodon optimization. Genes of prokaryotic origin have been expressed atvery high levels in transgenic chloroplasts (up to 47% of total solubleprotein, DeCosa et al., 2001). Both enzymes have similar catalyticactivity but the aphA-6 gene product has an extended ability to detoxifykanamycin and provides a wider spectrum of aminoglycosidedetoxification, including amikacin. The advantage of choosing kanamycinas a selectable marker is that it has no natural resistance, unlikespectinomycin resistance observed in most monocots or spontaneous pointmutation of the 16 S rRNA gene observed during the selection process. Inaddition, kanamycin is not in human clinical use as an antibiotic andseveral crops containing kanamycin resistant nuclear transgenes havebeen already approved by FDA for human consumption (e.g. flavor savortomatoes) and currently in the market place.

As shown in FIG. 24, in this non-limiting example, all transgenes areregulated by the plastid Prrn promoter; this 16S rRNA promoter drivesthe entire rRNA operon in the native chloroplast genome and containsbinding sites for both the nuclear encoded and plastid encoded RNApolymerases. Therefore, this promoter is capable of functioning in bothproplastids and chloroplasts (green and non-green, in the light anddark). The aphA-6 gene is further regulated by the gene 10 5′ UTRcapable of efficient translation in the dark, in proplastids present innon-green tissues (see GFP expression in proplastids of non-green cellsof corn and carrot in FIGS. 19 and 23 regulated by the 16S rRNA promoterand gene 10 UTR). The rps16 3′ UTR has been used to stabilize aphA-6gene transcripts. The aphA-2 (nptII) gene, on the other hand isregulated by the psbA promoter, 5′ and 3′ UTRs, which are lightregulated and highly efficient in the light, in chloroplasts (see A.Fernandez-San Millan, A. Mingeo-Castel, M. Miller and H. Daniell, 2003,A chloroplast transgenic approach to hyper-express and purify HumanSerum Albumin, a protein highly susceptible to proteolytic degradation.Plant Biotechnology Journal, in press; also see WO 01/72959). Therefore,a combination of both aphA-6 and aphA-2 genes, driven by regulatorysignals in the light and in the dark in both proplastids andchloroplasts, provides continuous protection for transformedplastids/chloroplasts around the clock from the selectable agent. Thegene(s) of interest with appropriate regulatory signals (gene X) areinserted downstream or upstream of the double barrel selectable system.Because multiple genes are inserted within spacer regions (DeCosa et al2001, Daniell & Dhingra, 2002), the number of transgenes inserted doesnot pose problems in transcription, transcript processing or translationof operons (WO 01/64024). In a variation of this example, aphA-6 andaphA-2 genes, coupled with different transgenes are inserted atdifferent spacer regions within the same chloroplast genome usingappropriate flanking sequences and introduced via co-transformation ofboth vectors.

Example 7 Genetic Engineering of the Corn and Ryegrass ChloroplastGenomes A. Transformation of Corn Chloroplast Genome

For genetic engineering of the corn chloroplast genome, corn specificsequences, flanking the targeted integration site in the cornchloroplast genome (trnI and trnA) were amplified with specific PCRprimers and subcloned to flank the betaine aldehyde dehydrogenase (BADH)selectable marker, and green fluorescent protein (GFP) reporter geneexpression cassette.

Callus cultures were initiated from aseptically excised immature zygoticembryos (1-2 mm in length), produced on self-pollinated ears of HiII(F1) maize plants. Ears were surface sterilized in a solution containing2.6% Sodium hypochlorite (prepared with commercial bleach) containing0.1% Tween 20 (polyoxyethylene sorbitan monolaurate) for 20 minutesunder continuous shaking, then rinsed 4 times in sterile distilledwater. The Embryos were then placed on the callus induction medium CI-1,which contained N6 salts and vitamins (463.0 mg/l(NH.sub.4).sub.2SO.sub.4, 2830.0 mg/lKNO.sub.3, 400 mg/lKH.sub.2PO.sub.4, 166.0 mg/l CaCl.sub.2, 185 mg/l MgSO.sub.4.7H.sub.2O,37.3 mg/l Na.sub.2-EDTA, 27.85 mg/l FeSO.sub.4.7H.sub.2O, 1.6 mg/lH.sub.3BO.sub.3, 4.4 mg/l MnSO.sub.4.H.sub.2O, 0.8, KI, 1.5 mg/lZnSO.sub.4.7H.sub.2O), 2% sucrose and 1.0 mg/l 2,4-D (2,4dichloro-phenoxy acetic acid), with the rounded scutellar side exposedand the flat plumule-radicle axis side in contact with the medium.Callus cultures were maintained in darkness at 25-28.degree. C. andsubcultured every two weeks.

Particle Bombardment of Embryogenic Calli

Micro projectiles were coated with DNA (pDA34-ZM-gfp-BADH andpDA33-ZM-aadA-BADH) and bombardment was carried out with the biolisticdevice PDS1000/He (Bio-Rad).

Prior to bombardment, embryogenic calli were selected, transferred oversterile filter paper (Watman No. 1), and placed on the surface of afresh medium in standard Petrti plates (100.times.15 mm). Gold particles(0.6 .mu.m) were then coated with plasmid DNA as follows: 50 .mu.l ofwashed gold particles were mixed with 10 .mu.l DNA (1 .mu.g/.mu.l), 50.mu.l of 2.5M CaCl.sub.2, 20 .mu.l of 0.1M spermidine and vortexed.Particles were cneterfuged for a few seconds at 3000 rpm and then theethanol was poured off. Ethanol washing was repeated five times, thenthe pellet was resuspended in 30 .mu.l of 100% ethanol and placed on iceuntil it was used for bombardment (the coated particles were used within2 hours). Bombardment was carried out with the biolistic device PDS1000/He (Bio Rad) by loading the target sample at level 2 in the samplechamber under a partial vacuum (28 inches Hg).

The callus cultures were bombarded with the maize chloroplasttransformation vectors using 1100 psi rupture discs. Followingbombardment, the explants were transferred to a fresh medium; plateswere sealed with micropore tape and incubated in darkness at25-28.degree. C.

Selection

Selection was initiated two days after bombardment. The bombarded calliwere transferred to callus induction medium containing 5-20 mM BA(betaine aldehyde) or 25-100 mg/l streptomycin. Selection was alsocarried out using 50-150 mM NaCl in combination with the BA to maintainosmostic pressure.

Regeneration

Regeneration was initiated 6 to 8 weeks after bombardment bytransferring the calli to a medium R1 containing Ms salts and vitaminssupplemented with 1.0 mg/l NAA (.alpha.-naphthalene acetic acid), 2%sucrose, 2 g/l myoinositol and 0.3% phytagel at pH 5.8. Regeneratedplants were transferred to R2 containing 1/2 MS salts and vitamins, 3%sucrose and 0.3% phytagel at pH 5.8. Regenerated plants were maintainedin light (16/8 hr photoperiod).

Shoot Multiplication The Surface Sterilization and Germination of CornSeeds

Corn seeds were surface sterilized in a solution containing 2.6% Sodiumhypochlorite (prepared from commercial bleach) containing 0.1% Tween 20for 20 minutes under continuos shaking, then rinsed four times insterile distilled water. Seeds were grown on MS medium at pH 5.8 indarkness. Nodal sections were excised aseptically from three day oldseedlings. The nodal sections appear as clear demarcations on thegerminated seedlings and represent the seventh node. When excised, thenodal cross sections are 1.3 to 1.5 mm in length.

Particle Bombardment of Nodal Sections

Prior to bombardment, 20-30 nodal sections were placed in the center ofeach petri plate with acropitila end up. Bombardment was carried outwith the maize chloroplast vectors, using 1100, 1300 and 1550 psirupture discs.

Multiple Shoot Induction and Selection

Nodal section explants are placed acropital end up on shootmultiplication medium SM1 composed of Ms salts and vitamins, 1.0 mg/l6BA (6-Benzyl amino purine), 3% sucrose and 5 g/l phytagel at pH 5.8under continuous light at 25.degree. C. Initiation of the shoot-tipclumps from the original shoot tips occurred 2 to 4 weeks after culture.Two days after bombardment, transformed nodal sections were transferredto shoot multiplication medium containing 5-20 mM BA or 50-100 mg/lstreptomycin selective agents. Subsequent subcultures at two weekintervals were carried out by selecting, dividing and subculturing greenclumps on selective shoot multiplication medium containing 5-20 mM BA or25-100 mg/l streptomycin.

Regeneration

The Multiple shoot clumps were regenerated by transferring them toregeneration medium M1 containing MS salts and Vitamins, 5 mg/l IBA and3% sucrose at pH 5.8. The developed shoots were regenerated bytransferring the shoot tip clumps to M2 medium containing 1/2 MS saltsand vitamins, 3% sucrose and 3 g/l phytagel at pH 5.8. It should befurther noted that all the regeneration media are supplemented with 5-20mM BA or 25-100 mg/l streptomycin as the selective agents.

To engineer the corn chloroplast genome free of antibiotic resistancegenes, maize calli were bombarded with a chloroplast expression vectorcontaining the green fluorescent protein (GFP) and the betaine aldehydedehydrogenase (BADH) genes as selectable or screenable markers. Tocompare the betaine aldehyde (BA) selection with streptomycin, anotherchloroplast expression vector was constructed containing the aada andthe BADH genes. The number of putative transgenic events was higher onBA selection than on streptomycin. Transgenic corn tissues screened onBA were examined using a laser-scanning confocal microscope. The GFPfluorescence was observed throughout the somatic embryos of corn.Chloroplast transformation of corn provides a suitable avenue for theproduction of edible vaccines and oral delivery of biopharmaceuticals.

Corn Chloroplast Transformation Vector

Corn chloroplast transformation vector facilitates the integration oftransgene into the inverted repeat (IR) region of the corn chloroplastgenome. The vector pLD-Corn-BADH contains the chimeric aadA gene and theBADH gene driven by the constitutive 16 S rRNA promoter and regulated bythe 3′ UTR region of psbA gene from petunia plastid genome. In thisconstruct both, aadA and BADH possess the chloroplast preferredribosomal binding site, GGAGG. Another vector used for corn chloroplasttransformation pLD-corn-UTR-BADH has the constitutive 16 S rRNA promoterdriving the expression of the dicistron, but BADH is under theregulation of the promoter and the 5′ UTR of the psbA gene and the 3′UTR of psbA gene, for enhanced expression. Since the expression of theforeign protein is desired in chromoplasts of corn seeds, the gene ofinterest needs to be under the control of a regulatory sequence that isfree from cellular control. In this context, examples of suitablecandidate regulatory sequences are the T7 gene 10-leader sequence andcry2Aa2 UTR. The T7 gene 10-leader sequence is used to express foreignproteins in transgenic chromoplasts. The cry2Aa2 UTR has been shown bythe inventor to accumulate as much foreign protein in chromoplasts asefficient as the psbA UTR in green tissues. Therefore the selectablemarker for additional vectors use the BADH gene under the regulation ofpsbA promoter and 5′UTR, as psbA is one of the most efficientlytranslated chloroplast genes in green tissues. When green tissue ornon-green embryogenic calli are used for introducing the transgene intothe corn chloroplast genome, it is preferred to use the light regulatedpsbA promoter/UTR or 16 S rRNA promoter/gene 10 UTR, respectively.

B. Ryegrass Chloroplast Transformation

Annual ryegrass chloroplast transformation vector facilitates theintegration of transgene into the inverted repeat (IR) region of theannual ryegrass chloroplast genome. The vector pLD-Ryegrass-BADHcontains the chimeric aadA gene and the BADH gene driven by theconstitutive 16 S rRNA promoter and regulated by the 3′ UTR region ofpsbA gene from petunia plastid genome. In this construct both, aadA andBADH possess the chloroplast preferred ribosomal binding site, GGAGG.Another vector used for ryegrass chloroplast transformationpLD-ryegrass-UTR-BADH has the constitutive 16 S rRNA promoter drivingthe expression of the dicistron, but BADH is under the regulation of thepromoter and the 5′ UTR of the psbA gene and the 3′ UTR of psbA gene,for enhanced expression. When green tissue or non-green embryogeniccalli are used for introducing the transgene into the corn chloroplastgenome, it is preferred to use the light regulated psbA promoter/UTR or16 S rRNA promoter/gene 10 UTR, respectively.

Vectors for Production of Edible Anthrax Vaccine in TransgenicChloroplast of Corn or Ryegrass:

Studies have confirmed the role of PA as the major protective antigen inthe humoral response but also indicate a significant contribution of LFand EF to immunoprotection. The LF amino terminal domain, amino acidresidues 1-254 (27 kDa) contains all the information necessary forbinding PA and mediating translocation, and this domain alone isnontoxic because the catalytic domain of LF, residues 255-776, isresponsible for lethality. Titers of antibody to both PA and LF frommice immunized with the combination were 4 to 5 times greater thantiters from mice immunized with either alone. Therefore we express theconstructs LF27-PA63 (PA63 is the cleaved active form of PA), CTB-LF27fusion proteins, and LF27 and PA independently within the same edibleplant as a standard. The LF27-PA63 and CTB-LF27 constructs are expressedalone and together as an operon in corn and ryegrass. It has beendemonstrated that Rotavirus enterotoxin proteins fused with CTB isprocessed via the MHC II pathway generating a strong T-cell response.Thus CTB-fusion proteins produced in plants are ideal for oral delivery.By expressing the CTB-LF27 and PA-LF27 we maximize immunity to lethaltoxin challenge. This is because both Gm1 ganglioside and anthrax toxinreceptor (ATR) can be bound by ligands and work synergistically formaximum immune response. A flexible hinge was introduced between fusionproteins to reduce steric hindrance. Specifically aglycine-proline-glycine-proline hinge between CTB-LF27 andproline-glycine-proline-glycine hinge between LF27-PA63 was used. Theapplication of less frequently used codons in plants within the hingepeptide promotes translational arrest during the protein elongationprocess, facilitating subunit folding prior to translation. Theefficiency of folding of some proteins is increased by controlled ratesof translation in vivo.

Chloroplast Transformation Protocol for Corn and Ryegrass:

Using either immature embryos (IEs) or embryogenic callus derived fromIEs as a target for biolistic gene transfer is a well-establishedprocedure for stable integration into the nuclear genome of corn orryegrass. For biolistic transfer of integrative chloroplast expressionvectors, the gene transfer protocol is adjusted and smaller particlesizes (0.6 .mu.m diameter) are used. Microprojectiles are coated withplasmid DNA (chloroplast vectors) and bombardments are carried out withthe biolistic device PDS 1000/He (Bio-Rad) as is well-known in the artrelating to the use of the “gene-gun.” Expression levels fromchloroplast regulatory sequences and the size of the proplastids arelimiting factors for the successful chloroplast transformation usingnon-green, embryogenic callus tissues as a target for the gene transfer.Therefore, it is most desirable, when using the present invention withplant species not tested here, to compare green shoot meristematiccultures with non-green embryogenic callus as target tissue forchloroplast transformation. Protocols for the establishment of thesetissue types are reported for corn and the grasses and are establishedin the Alpeters laboratory at University of Florida at Gainesville forryegrass.

The timing of gene transfer after culture initiation and the durationand level of selection affect transgenic events while reducing thenumber of chimeric plants and achieving homoplasmy and are bestevaluated empirically. BADH and aadA selectable markers are comparedwith the corresponding selective agents. Selection is to be maintainedduring the regeneration process of plants. Regenerated plants are thenanalyzed by PCR and Southern blot for integration in the corn orryegrass plastome.

PCR is done using DNA isolated from control and transgenic plants inorder to distinguish a) true chloroplast transformants from mutants andb) chloroplast transformants from nuclear transformants. In order totest chloroplast integration of the transgenes, the 3′ primer willanneal to the selectable marker gene while the 5′ primer will anneal tothe native chloroplast genome. No PCR product is expected with nucleartransgenic plants or mutants using this set of primers. This screeningis essential to eliminate mutants and nuclear transformants. Total DNAfrom wildtype and transgenic plants is isolated and used as a templatefor PCR reactions. Southern blots allow one skilled in the art todetermine the copy number of the introduced foreign gene per cell aswell as to test homoplasmy. There are several thousand copies of thechloroplast genome present in each plant cell. When foreign genes areinserted into the chloroplast genome, not all chloroplasts willintegrate foreign DNA resulting in heteroplasmy. To ensure that only thetransformed genome exists in transgenic plants (homoplasmy), theselection process is continued. In order to confirm homoplasmy at theend of the selection cycle, total DNA from transgenic plants is probedwith the chloroplast border (flanking) sequences (the trnI-trnAfragment). Wild type fragment size is observed along with the largerfragments of transformed plastomes. Presence of a large fragment (due toinsertion of foreign genes within the flanking sequences) and absence ofthe native small fragment confirms homoplasmy. The copy number of theintegrated gene is determined by establishing homoplasmy for thetransgenic chloroplast genome.

Generate Transgenic Ryegrass and Corn Plants Expressing an EdibleAnthrax Vaccine and Characterize Transgene Integration and Expression

Using the aforementioned transformation protocols, vectors for theproduction of an orally administrable form of PA are introduced inryegrass and corn plants. Site specific vector integration into theryegrass or corn plastome is then confirmed by PCR and Southern blotanalysis as specified. Western blot verification of PA verifies thatrecombinant anthrax protective antigen proteins are antigenicallysimilar to native PA using monoclonal antibodies against PA (AdvancedImmunoChemical G1-Ba1). PA is quantified by a ELISA using purified PAantigen as standard and commercially available antibody. Electronmicroscopy is next carried out in mature leaves of chloroplast or matureseeds amyloplasts of transgenic plants to detect inclusion bodiesaccording a protocol and similar to several published electronmicrographs of transgenic chloroplasts, with immunogold label of foreignproteins. The PA protein is then purified using a two step protocol,such as that described in Ahuja, N., Kumar, P., & Bhatnagar, R. (2001),Rapid Purification of Recombinant Anthrax-Protective Antigen underNondenaturing Conditions, Biochemical and Biophysical ResearchCommunications, 286, 6-11. The protein is purified on AKTA-FPLC usinganion exchange Resource Q column (Pharmacia). The protein is then elutedfrom the column with a 20 ml decreasing gradient of ammonium sulphate.Fractions of 1 ml each are collected, analyzed on SDS-PAGE, and thosecontaining PA are pooled. With an affinity tag, the PA protein canoptionally be purified using metal-chelate affinity chromatography underdenaturing conditions. Ten ml of each fraction is then analyzed on 12%SDS-PAGE. Fractions containing the protein are collected, pooled, anddialyzed against 10 mM Hepes buffer containing 50 mM NaCl and storedfrozen at −70.degree. C. in suitable aliquots.

Recombinant PA proteins are then assayed for their functional activityin the J774A1 (American Type Culture Collection) macrophage lysis assay.Varying concentrations of PA protein along with LF (1 mg/ml) are addedto the cells. The native PA along with LF is kept as the positivecontrol. After 3 h, cell viability is determined using the MTT(3-(4,5-dimethyl thiazol-2-yl),-5-diphenyltetrazolium bromide) dye andthe resulting precipitate is dissolved in a buffer containing 0.5% (w/v)sodium dodecyl sulfate, 25 mM HCl in 90% isopropyl alcohol. Absorptionat 540 nm is measured and percent viability determined.

PA can be tested for susceptibility to cleavage by trypsin. To do so,the PA protein (1.0 mg/ml) is incubated with trypsin (1 ng/mg ofprotein) for 30 min at room temperature in 25 mM Hepes, 1 mM CaCl₂, 0.5mM EDTA, pH 7.5. The digestion reaction is stopped by adding PMSF to aconcentration of 1 mM. Trypsin nicked PA (1.0 mg/ml) is incubated withLF (1.0 mg/ml) and in 25 mM Tris, pH 9.0, containing 2 mg/ml CHAPS(3-{(3-cholamidopropyl) dimethyl ammonio}-propanesulfonic acid) for 15min at room temperature. Samples are applied to nondenaturing 4.5%polyacrylamide gel.

The binding of PA protein to cell surface receptor is analyzed in 24well plates using constant amount of radio-iodinated native PA (0.1mg/ml). J774A.1 (ATCC) cells are washed twice with cold HBSS for 5minutes each time and then placed on ice. The medium is replaced withcold binding medium (DMEM, Dulbecco's Modified Eagle Medium, withoutsodium bicarbonate containing 1% bovine serum albumin and 25 mM, Hepes,pH 7.4). The cells are incubated with 0.1 mg/ml of iodinated PA andvarying concentrations of the recombinant PA protein at 4.degree. C. for3 h and then washed with cold HBSS. The cells are then dissolved in 0.1N NaOH and radioactivity measured in Gamma counter.

Leaves from transgenic lines producing epitope tagged products arefrozen and powdered at 4.degree. C. using a microdismembranator andproteins are extracted in PBS with 1% Triton X-100. Fusion proteins arepurified by affinity chromatography on a nickel-agarose bed, usingstandard 6-His methods, as described above.

Assessment of Immunogenic Properties of Transgenic Plant-Derived PA:

Corn and ryegrass expressing PA as potential edible vaccines againstanthrax are characterized using the protocol described above. These arethen evaluated for the ability of PA-expressing corn seeds or corn orryegrass leaves or bay to function as edible vaccines for the inductionof serum and mucosal (bronchial lavage, nasal, vaginal, and fecal)antibodies by ELISA. Antibodies induced by feeding the transgenic cornor ryegrass to mammals neutralize the biologic activity of anthraxlethal toxin. This activity can be confirmed in an in vitro macrophagecytotoxicity assay. Antibody responses in mice and humans followingingestion of transgenic potatoes and corn expressing recombinantbacterial proteins have been successfully demonstrated.

Oral immunization of mice and other mammals by feeding transgenic plantsor plant parts is accomplished as follows. In the case of corn andryegrass, female BALB/c mice are fed transgenic corn or ryegrass,control corn or ryegrass, or soluble rPA in conjunction with the mucosaladjuvant LT(R192G). The amount of rPA fed to control animals is basedupon the amount of PA in the transgenic corn or ryegrass fed to theanimals. That amount correlates with the amount of transgenic corn orryegrass a mouse will consume in a one hour period. Mice tend to eatgrass if a small amount of vanilla extract is placed on each leaf. Twoadditional groups can be included in which the mucosal adjuvantLT(R192G) is administered in conjunction with the transgenic or controlcorn or ryegrass. Edible vaccines administered to mice often require thepresence of a mucosal adjuvant due to the small amount of material thatcan be consumed by a mouse. However, this is not necessary when usingthe plants of the present invention to vaccinate humans, or other largemammals due to the volume which can be consumed by the animal.Twenty-five micrograms of the adjuvant should be applied directly to thecorn or ryegrass before consumption when testing mice.

Intranasal immunization is accomplished in mice as follows. Mice arefirst lightly anesthetized with Isoflurane for approximately 45 seconds.The immunizing inoculum (5-10 ml per animal/per dose) is deliveredintranasally to the external nares of one nostril with a pipette tip.

Oral immunization: Oral inoculations consisted of 500 ml of the antigenpreparation in saline delivered intragastrically with a blunt-tipfeeding needle (Popper & Sons, Inc.).

Sample collection: Animals are sacrificed following euthanasia byCO.sub.2 inhalation. Blood is collected by cardiac puncture and theserum is separated in Microtainer tubes. Bronchoalveolar lavages (BAL)are obtained by inserting a 20 G cannula in the exposed trachea andinjecting 1 ml of PBS supplemented with protease inhibitors. The bufferis allowed to bathe the lung for approximately 20 seconds and then it issuctioned out; this procedure is repeated three times in each mouse. Theresulting BAL fluid is immediately centrifuged (400 g, 2 min, 4.degree.C.) and the supernatant is saved. To obtain nasal lavages a flexible 24gauge canula is inserted into the posterior opening of the nasopharynxand a total of 150 ml ml PBS+ protease inhibitor is injected into theopening. The outflow is collected as the nasal wash. Vaginal washes areobtained by washing the vaginal mucosa three times with 50 .mu.l of PBScontaining 0.01% NaN.sub.3. For determination of fecal IgA, feces arecollected and frozen overnight at −70.degree. C., lyophilized,resuspended in 800 .mu.l PBS containing 0.05% sodium azide per 15 fecalpellets, centrifuged at 1,400.times.g for 5 minutes, and the supernatantstored at −20.degree. C. until assayed.

Evaluation of humoral and mucosal antibodies: Each serum, BAL, nasalwash, vaginal wash, and fecal extract sample is individually analyzed byELISA. For all ELISA assays, 96-well plates are coated with 500 ng perwell of rPA and incubated overnight at 4.degree. C. All subsequent stepsare carried out at room temperature. After blocking with 1% BSA, twofoldserial dilutions of serum, BAL, nasal wash, vaginal wash, or fecalextract from the experimental animals are added. Alkaline phosphataseconjugated rabbit anti-mouse IgG or anti-mouse IgA are used fordetermination of total IgG or IgA. Biotinylated anti-mouse IgG1, IgG2a,IgG2b or IgG3 followed by alkaline phosphatase conjugated streptavidinare used to quantify antibody isotypes. Optical density at 405 nm isdetermined using an ELISA reader.

REFERENCES

The following references, along with all other references mentionedherein, and patent applications to which this application may claimpriority, are incorporated herein by reference in their entirety.

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1-25. (canceled)
 26. An orally-administrable vaccine compositioncomprising a chloroplast expressed protective antigen for conferringimmunity to said antigen in a mammal, said vaccine comprising edibleplant material.
 27. The vaccine composition of claim 26, wherein saidantigen confers an immunoprotective response in said mammal.
 28. Thevaccine composition of claim 26, wherein said plant material comprises achloroplast expression vector encoding said antigen, said antigen beingfused to a sequence encoding cholera toxin B (CTB), said CTB forming CTBpentamers which exhibit intact transcytosis to the external basolateralmembrane of intestinal epithelium.
 29. The vaccine composition of claim26, wherein said vaccine composition is heat-stable.
 30. The vaccinecomposition of claim 26, wherein said plant is selected from the groupconsisting of tomato, carrot, low nicotine tobacco, corn, and ryegrass.31. A method for inducing an immune response against an antigen,comprising feeding to said mammal an effective amount of the vaccinecomposition of claim
 1. 32. An orally-administrable vaccine compositionas claimed in claim 1, for conferring immunity to Yershina pestis to amammal, said vaccine comprising plant material comprising V and F1antigens of Y. pestis.
 33. The vaccine composition of claim 32, whereinsaid plant material comprises a plant plastid genome having a geneticsequence encoding for V and F1 antigens of Y. pestis.
 34. A process forvaccinating a mammal against Yershina pestis comprising feeding to saidmammal an effective amount of the vaccine composition of claim
 32. 35.An orally-administrable vaccine composition as claimed in claim 1 forconferring immunity to Bacillus anthracia to a mammal, said vaccinecomprising plant material comprising anthrax protective antigen.
 36. Thecomposition of claim 35, wherein said vaccine is free of both anthraxedema factor and anthrax lethal factor.